A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues

The ras genes play an important role in the development and progression of human tumours. Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling. In this study, we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours.

R E S E A R C H A R T I C L E Open Access

A novel anti-p21Ras scFv antibody reacting

specifically with human tumour cell lines

and primary tumour tissues

Ju-Lun Yang1*, Du-Xian Liu2, Shi-Jian Zhen2, Yun-Gang Zhou2, Dai-Jun Zhang1, Li-Ying Yang1, Hao-Bing Chen1 and Qiang Feng1

Abstract

Background: The ras genes play an important role in the development and progression of human tumours

Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling In this study,

we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours

Methods: The coding sequences of H-ras, K-ras, and N-ras were separately ligated into the vector pET-28a(+)

Then, recombinant expressing plasmids were induced by IPTG for p21Ras expression in E coli Hybridoma cell lines producing anti-p21Ras monoclonal antibodies were isolated using wildtype p21Ras proteins as immunogens Anti-p21Ras scFv antibody was prepared from the hybridoma by the phage scFv display method The immunoreactivity

of the anti-p21Ras monoclonal antibody and the scFv antibody was identified by ELISA and immunocytochemistry Results: We prokaryotically expressed wildtype H-p21Ras, K-p21Ras and N-p21Ras and generated the hybridoma cell line KGH-R1, producing anti-p21Ras monoclonal antibodies It was demonstrated that KGH-R1 monoclonal

antibody could recognize wildtype and mutated H-p21Ras, K-p21Ras and N-p21Ras in human tumour cell lines

In all 14 types of primary human cancer tissues tested, the monoclonal antibody presented strong immunoreactivity but showed weak or negative immunoreactivity in the corresponding normal tissues Subsequently, we prepared anti-p21Ras scFv from hybridoma KGH-R1, which showed the same immunoreactivity as the original

monoclonal antibody Sequence analysis demonstrated that the nucleotides and amino acids of the scFv exhibited

an approximately 50 % difference from the anti-p21Ras scFv reported previously

Conclusions: This study presents a novel anti-p21Ras scFv antibody Our data suggest that the scFv may be useful for ras signalling blockage and may be a potential therapeutic antibody for ras-derived tumours

Keywords: p21Ras, scFv, Tumour, Immunoreactivity, Monoclonal antibody

Background

Because of the important role of ras in carcinogenesis and

progression, the ras signalling pathway has attracted

con-siderable attention as a target for anticancer therapy The

ras gene product, p21Ras, is a monomeric

membrane-localized G protein of 21 kD, which functions as a

molecu-lar switch converting signals from the cell membrane to the

nucleus and linking receptor and nonreceptor tyrosine

kinase activation to downstream cytoplasmic or nuclear events The biological effects of p21Ras depend on its bio-chemical properties of being a small GTP-binding protein and on its correct cellular location at the cytoplasmic face

of the plasma membrane [1] Thus, the neutralization of p21Ras proteins in the cytoplasm using specific antibodies may block ras signalling and constitute a promising thera-peutic strategy [2]

It is well known that whole antibodies can penetrate cells only with difficulty due to their large molecular size

In recent years, a series of low-molecular-weight anti-bodies containing antigen-binding domains have been

* Correspondence: yangjulun@sina.com

1 Department of Pathology, Kunming General Hospital/Kunming Medical

University, Kunming 650032, Yunnan Province, China

Full list of author information is available at the end of the article

© 2016 Yang et al Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

explored to develop antibody-based drugs with better

tumour penetration, such as antigen-binding fragment [3],

single chain fragment variable (scFv) [4], and single-domain

antibodies [5] It has been found that scFv antibodies

pene-trate the cell membrane better than whole antibodies [6, 7]

and result in no immunological rejections due to lacking

the Fc fragment [8, 9], giving them advantages as

intracellu-lar immunization and therapeutic antibodies Currently,

scFv antibodies have been applied in many fields, including

anti-viral and cancer therapy [10–12]

Both overexpression and mutation can activate ras

genes The overexpression of p21Ras has been detected

in many human tumours [13–17] The overexpression of

ras family members led to the acquired resistance of

cancer to cetuximab treatment [18] It has been found

that ras mutations are present in approximately 33 % of

all human tumours [19] K-ras mutations occur

fre-quently in non-small-cell lung, colorectal, and pancreatic

carcinomas; H-ras mutations are common in bladder,

kidney, and thyroid carcinomas; and N-ras mutations

are found in melanoma, hepatocellular carcinoma, and

haematologic malignancies [20] However, previously

re-ported anti-p21Ras antibodies were derived from mutated

p21Ras antigen [21–23] In this study, we isolated

hybrid-oma cell lines producing p21Ras monoclonal

anti-bodies, using wildtype p21Ras proteins as immunogens,

prepared anti-p21Ras scFv antibodies from the

hybrid-omas, and then investigated their immunoreactivity with

human tumour cell lines and primary tumour tissues

Methods

Preparation of the wildtype p21Ras proteins

The coding sequences (CDS) of the H-ras, K-ras, and

N-ras genes were chemically synthetized according to their

wildtype mRNA sequences published in NCBI GenBank:

BC005219 for N-ras The restriction enzyme Bam HI

cutting site GGATCC was ligated at the 5′ end of the

CDS, and the Hind III cutting site AAGCTT was ligated

at the 3′ end during synthesis After digestion with Bam

HI and Hind III, the three CDS fragments were ligated

separately into the vector pET-28a(+) by T4 ligase Then,

recombinant expressing plasmids were transformed into

E coli BL21(DE3) and screened by kanamycin, induced

by IPTG for p21Ras expression [24] Expressed p21Ras

proteins were purified by Ni2+-NTA resin with the mild

denaturant urea and then underwent SDS-PAGE

ana-lysis, followed by dialysis for renaturation

Preparation of hybridomas producing broad-spectrum

anti-p21 Ras mAb

Balb/c mice were immunized by injection with wildtype

H-p21Ras expressed prokaryotically The mouse splenic

B lymphocytes were fused with myeloma cell lines SP2/

0 After selective culture using HAT selective culture medium, the fused hybridoma cells were screened by an in-direct ELISA method with all three wildtype p21Ras pro-teins and then cloned and subcloned to obtain hybridoma cell lines producing monoclonal antibodies against wildtype H-p21Ras, K-p21Ras and N-p21Ras All hybridoma cell lines were subcloned twice Finally, the hybridoma cell lines were injected into the peritoneal cavity of Balb/c mice to produce monoclonal antibodies [21] A completed ARRIVE guidelines checklist is included in Additional file 1 Human cancer cell lines were used to identify the immunoreactivity

of the monoclonal antibodies by Western blot Primary tumour tissues and their corresponding normal tissues were employed to investigate the tumour reactivities of the monoclonal antibodies by immunohistochemical staining, and the results were described by HSCOREs [25]

Construction of scFv phage display library

Hybridoma cell line KGH-R1, which produced excellent broad-spectrum anti-p21Ras mAb, was used to con-struct a phage scFv display library Total RNA was iso-lated from KGH-R1 cells using RNAiso plus (MrcGene) and reverse transcribed to cDNA using the RevertAid™

H Minus First Strand cDNA Synthesis Kit (Fermentas)

variable region (VL) were amplified using primers pro-vided by the Recombinant Phage Antibody System (GE Healthcare, RPAS) A DNA linker complementary to the

amplification to construct the scFv gene

The phage scFv display library was constructed accord-ing to the RPAS system manual (GE Healthcare) Briefly, the scFv fragment genes and the phagemid pCANTAB-5E were sequentially digested with the Sfi I and Not I restric-tion enzymes After purificarestric-tion by agarose gel extracrestric-tion, both the scFvs fragments and the pCANTAB-5E vector with Sfi I and Not were ligated using T4 DNA ligase, transformed into competent E coli TG1 cells, and

ampicillin and 2 % glucose) at 37 °C All of the bacterial single colonies that grew on the SOBAG plate were col-lected, mixed and cultured in 2 × YTAG medium The helper phage M13K07 at 1011pfu was added to the collec-tions and co-cultured at 30 °C for 1 h to obtain recombin-ant phages expressing the scFv recombin-antibodies and g3p fusion proteins on the surface The scFv phages were purified and concentrated by polyethylene glycol (20 % PEG8000, 2.5 mol/L NaCl) The pellet was resuspended in PBS to obtain the phage scFv display library

Enrichment of scFv phage

The phage scFv display library underwent three rounds

of panning using H-p21Ras, K-p21Ras and N-p21Ras

protein antigens, respectively Briefly, the flask was coated

blocking with BSA, the library was added to the flask and

incubated at 37 °C for 2 h, followed by washing with PBST

(containing 0.05 % tween-20, pH 7.4) 10 times, followed

by washing PBS (without Tween-20) an additional 10

times to remove unbound phage particles Specifically

bound phages were eluted with 0.1 M HCL/glycine

(containing 0.1 % BSA, pH 2.2) The eluent was

neu-tralized with pH 9.0 Tris/HCl, was used to infect

TG1 cells and was then rescued with helper phage

M13K07 These enriched scFv phages were used in

the next round of panning [26, 27] In each panning

round, the numbers of input and output phages were

counted using the double agar layer method

Identification of scFv-phages

E coli TG1 cells were infected by the final enriched phage

pools and cultured on an SOB-AG plate Single colonies

were picked and rescued individually with M13K07 in

96-well plates The phages from individual 96-wells were assayed

by ELISA to detect their specific antigen binding activities,

utilizing p21Ras proteins as antigens, scFv-phage as

pri-mary antibody, and HRP-conjugated mouse anti-M13

mAb as second antibody The positive phages were further

identified by Sfi I and Not I enzyme digestion and PCR

amplification with pCANTAB-5E primers (forward,

S1: 5-CAACGTGAAAAAATTATTATTCGC-3; reverse,

S6: 5-GTAAATGAATTTTC TGTATGAGG-3) The

amp-lified products were ligated into a pMD18-T vector for

se-quence analysis

Bioinformatics analysis of scFv gene and 3D modelling

The sequence of the scFv gene was blasted with known

murine genes for homology analysis in the GenBank

database, as described previously [28] The amino acid

residues, CDRs and FRs were determined according to

the IMGT numbering system and Kabat by IgBLAST

[28] The 3D structure of scFv antibody was generated

by SWISS-MODEL [29, 30]

Expression of soluble scFv antibodies

The expression of soluble scFv antibodies was performed

according to the manual and reference [31] Briefly, the

p21Ras-positve phages were used to infect HB2151 cells

and cultured in plates A single colony was picked and

grown overnight to express soluble anti-p21Ras scFv

antibodies with E-tag peptide at the end through the

induction of IPTG The soluble scFv antibodies were

extracted from the HB2151 culture and concentrated by

PEG8000/NaCl

Immunocytochemistry/immunohistochemistry and Western blot

Human tumour cell lines harbouring wildtype or mutated Ras [32, 33], primary tumour tissues and the corresponding normal tissues were employed to detect the immunoreac-tivities of the scFv antibodies by immunohistochemical staining The soluble scFv antibodies were used as the pri-mary antibody, whereas Anti-E-tag monoclonal antibody conjugated with HRP served as the second antibody to bind E-tag protein ligated to the end of the scFv antibody The scFv immunoreactivity with p21Ras was further detected

by Western blot in the tumour cell lines MDA-MB-435, MDA-MB-231 and SKOV3 (ATCC USA) and the normal cell line KMB17 (normal human embryonic diploid lung fibroblast cell, constructed by the Institute of Medical

was used as a control, with β-actin monoclonal anti-body conjugated with HRP

Ethics statement

This study including animal work has the approval of the Ethics Board of Kunming General Hospital and is also in accordance with the Helsinki Declaration of 1975 Written informed consent was obtained from every patient All human tissues were processed anonymously

Results

Preparation of hybridomas and anti-p21Ras monoclonal antibodies

By cloning the CDS of the H-ras, K-ras, N-ras genes into pET-28(+) vectors, we constructed a prokaryotically expressive vector (Fig 1a, b) that effectively expressed H-p21Ras, K-p21Ras and N-p21Ras protein in E coli BL21 SDS-PAGE analysis showed that the apparent molecular weight of the three p21Ras proteins was 25 KD (Fig 1c) The purity of p21Ras protein was more than 95 % H-p21Ras was used as an immunogen to immunize BALB/c mice to isolate hybridomas To achieve mono-clonal antibodies that simultaneously recognized H-p21Ras, K-p21Ras and N-H-p21Ras, the three p21Ras were used as antigens to screen the hybridomas in an ELISA assay In total, we obtained 13 hybridomas that pro-duced monoclonal antibodies against the three different p21Ras proteins Karyotype analysis showed that the me-dian chromosome numbers of the 13 hybridomas were 97–100 Immunochromatography indicated that the monoclonal antibodies produced by all 13 hybridomas were of the IgG2b/κ subtype Monoclonal antibodies in ascites reached a high titre of 1:128000 The immunoreac-tivities of the monoclonal antibody KGH-R1 were deter-mined by Western blot and immunohistochemistry The KGH-R1 monoclonal antibody demonstrated positive im-munoreactivities in all 13 human solid tumour cell lines and 2/6 leukaemia cell lines (Table 1) In all 14 types of

primary human cancer tissues tested here, the monoclonal

antibody presented strong immunoreactivity but showed

weak or negative immunoreactivity in the corresponding

normal tissues (Table 2, Fig 1d)

Construction and panning of scFv phage display library

The scFv gene consists of a heavy chain variable region

(VH) and a light chain variable region (VL), joined by a

Then, we connected both to DNA linker by overlapping

extended PCR to construct a 750 bp scFv gene (Fig 2b)

The scFv gene repertoire was ligated to the phagemid

pCANTAB 5E and then transformed into competent

TG1 colonies transformed by the recombinant phage

plasmids (Fig 2c) We collected all of the TG1 colonies,

co-cultured them with M13KO7 helper phages, and

established the scFv phage display library, in which

scFv-g3p fusion proteins were expressed at the tips of the

phages To obtain phages expressing anti-p21Ras scFv

antibodies from the library, the library was selected and panned using the H-p21Ras, K-p21Ras, and N-p21Ras proteins, in turn After three rounds of panning, the eluted

pFU/mL (Fig 2d)

Characteristics of anti-p21Ras scFv antibodies

To obtain monoclonal phages expressing anti-p21Ras scFv antibodies, we infected TG1 cells with the enriched scFv phage pool and plated them on SOBAG plates Then, 40 colonies were rescued by M13K07 Indirect ELISA tests showed that all 40 colonies expressed scFv antibodies that could bind H-p21Ras, K-p21Ras and N-p21Ras simultaneously Recombinant phagemids were iso-lated from each of the 40 colonies and digested by Sfi I and Not I and then ligated with pMD18-T vector for DNA sequencing Sequence analysis demonstrated that the se-quences of the 40 colonies were the same The number of amino acids and complementary determining regions

Fig 1 Wildtype p21Ras proteins and anti-p21Ras monoclonal antibodies a The plasmid map of recombinant pET-28a(+) vector expressing H-ras,

in which H-ras gene CDS with Bam HI and Hind III restriction enzyme sites was cloned into a pET-28a(+) vector with a histidine tag-compatible end b The three recombinant pET28a(+) plasmids were digested into two fragments by Bam HI and Hind III, the 570 bp of the ras gene CDS and

5340 bp of the pET-28a(+) vector c SDS –PAGE analysis showed that the molecular weight of the three p21Ras proteins with His tag was 25 kDa.

d KGH-R1 monoclonal antibody against three p21Ras proteins demonstrated strong immunoreactivity to cancer tissues but negative immunoreactivity

to corresponding normal tissues

IgBLAST KGHR1-scFv had 738 nucleotides encoding 246

VL consisted of 114 amino acids, and the flexible amino acid linker consisted of 15 amino acids Both the VH and

VLfragments had 3 CDR regions and 4 FR regions (Fig 3a)

subfamily III and that VL belongs to the subfamily VIIκ chains We constructed a three-dimensional model of the scFv antibody by the SWISS MODEL method In the 3D

connected by a polypeptide linker chain (Fig 3b)

Immunoreactivity of soluble anti-p21Ras scFv antibodies

Soluble anti-p21Ras scFv antibodies were prepared by infecting the non-amber suppressor E coli strain HB2151 with the recombinant phages The Western blot assay showed that anti-p21Ras scFv antibody could spe-cifically bind the p21Ras protein in the tested cells (Fig 3c) We further evaluated the immunoreactivities of scFv in human tumour cell lines, primary solid tumour tissues and normal tissues by immunohistochemical staining The results demonstrated that the soluble scFv displayed strong immunostaining in all the human tumour cell lines and all types of primary human cancer tissues tested here However, all of the corresponding normal tissues showed negative immunostaining The granular positive signal was mainly located in the cyto-plasm and membrane (Table 3, Fig 3d) The results indi-cated that the obtained scFv could recognize the p21Ras antigen epitope in tumour cells

Discussion

Antibody-based drugs are promising agents for cancer therapy In recent years, certain antibody drugs have been clinically approved and used frequently in cancer treatment Trastuzumab, a chimeric monoclonal anti-body against Her2 protein, is the most impressive target drug for breast cancer therapy [34, 35] Rituximab, an anti-CD20 monoclonal antibody, is another of the most effective drugs in B cell malignant lymphoma treatment [36] The gene ras is a well-known and important onco-gene involved in the development and progression of many human tumours, but no antibody drugs targeting the ras gene have been applied clinically The main rea-son is that the intracellular location of p21Ras has lim-ited the effects of antibodies because intact antibodies can not penetrate the cell membrane Thus, small mo-lecular antibodies, such as scFv, are proposed to treat ras-derived tumours

Because hybridoma cell lines are the most important resources for scFv [37–39], we established anti-p21Ras hybridomas using prokaryotically expressed wildtype H-p21Ras protein as an immunogen H-H-p21Ras, K-H-p21Ras and N-p21Ras were used to screen the hybridoma clones

Table 2 Immunoreactivity of KGH-R1 mAb in human tumours

and corresponding normal tissues

Primary tumour tissues

Normal tissues Colorectal cancer 30 208.69 ± 84.40 6.88 ± 1.53

Oesophageal cancer 30 163.73 ± 66.00 8.50 ± 3.28

Lung adenocarcinoma 30 238.63 ± 72.00 7.00 ± 1.77

Lung squamous cell carcinoma 30 178.26 ± 83.99 7.00 ± 1.77

Lung small cell carcinoma 30 134.38 ± 91.58 7.00 ± 1.77

Papillary thyroid carcinoma 30 239.33 ± 75.51 8.43 ± 2.32

Hepatocarcinoma 30 184.38 ± 96.75 11.29 ± 5.51

Prostate carcinoma 30 114.77 ± 70.59 13.00 ± 4.24

Endometrial cancer 30 139.29 ± 86.00 15.75 ± 5.72

Renal cell carcinoma 30 117.50 ± 73.86 16.00 ± 5.85

a

Mean ± SD

Table 1 Immunoreactivity of KGH-R1 mAb in human tumour

cell lines

Tumour cell lines a Ras status b Western blot Immunocytochemistry

-a

QGY7703, SMMC7721, HepG2: hepatocarcinoma; BGC853, MKN28: gastric

cancer; HCT116: colorectal cancer; T24: bladder cancer; SKOV3: ovary cancer;

MDA-MB-231, MDA-MB-435, MCF7: breast cancer; HeLa: cervical cancer; Hep2:

laryngocarcinoma; C8166, K562, Daud I, HL60, MT4, THP1: leukaemia.

b

published data

Finally, the hybridoma KGH-R1, producing a

broad-spectrum monoclonal antibody against the three p21Ras

proteins, was isolated Interestingly, the monoclonal

antibodies reacted with both wildtype and mutated

p21Ras in human tumour cell lines and presented

excel-lent immunoreactivities with the majority of human

tumour cell lines and primary tumour tissues but

nega-tive immunostaining in the corresponding normal

tis-sues The characteristics mentioned above give these

antibodies important value as therapeutic prodrugs To

our knowledge, this study is the first to use wildtype p21

Ras as an immunogen to prepare monoclonal antibodies

The anti-p21Ras monoclonal antibodies reported

previ-ously were derived from mutated p21Ras, of which

Y13-259 and Y13-238 were generated using p21 protein

encoded by the v-ras gene of the Harvey murine sarcoma

virus as an immunogen [22] RAP-1 ~ 5 were generated

utilizing a synthetic H-p21Ras peptide reflecting amino

acid positions 10–17 with a mutation at codon 12 as the

immunogen [23] RASK1 ~ 16 were generated using

mu-tated K-p21Ras from Kirsten murine sarcoma virus as the

immunogen [21]

Antibody genes can be fused into phage genes, and

then the antibodies can be expressed and displayed on

the surface of the phages as fusion proteins [40] The

phage display library has thus become the most popular

technique for constructing and selecting scFv antibodies

from hybridomas or B lymphocytes due to its benefits,

such as the high diversity of the antigenic repertoire and the ability to rapidly select specific antibodies [41] In this study, we employed the phage display library method to isolate scFv antibodies against human p21Ras proteins Fortunately, we obtained scFv antibodies that maintained all of the immunoreactivities of the original monoclonal KGH-R1 antibodies, including the recogni-tion of wildtype and mutated p21Ras proteins, reacrecogni-tion with H-p21Ras, K-p21Ras and N-p21Ras, and strong im-munostaining in tumour cell lines and primary tumours These results showed that scFv is useful for ras signal-ling blockage and may be a therapeutic antibody for ras-derived tumours

The scFv antibodies presented here were dramatically different from previous anti-p21Ras scFv in both their nucleotide sequences and their amino acid sequences [42, 43] There was a 49.8 % difference from Y13-259 scFv and a 53.73 % difference from Y13-238 scFv, mostly located in the CDRs (Fig 3a) Our data indicated that KGH-R1 scFv recognizes different antigen epitopes, based on a comparison of Y13-259 scFv and Y13-238 scFv, and it is a novel anti-p21Ras scFv antibody

Conclusions

In conclusion, this study presents a novel anti-p21Ras scFv antibody, KGH-R1 scFv, produced by hybridoma and phage scFv display library methods Sequence analysis showed that KGH-R1 scFv antibody has an approximately

Fig 2 Preparation of anti p21Ras scFv a Schematic diagram of scFv The variable region of the heavy chain (V H ) and the variable region of the light chain (V L ) were combined with a polypeptide linker b Construction of scFv gene The 340 bp V H fragment and 325 bp V L fragment were amplified from the cDNA of the KGH-R1 hybridoma This assembly reaction ultimately produces 750 bp of scFv c The recombinant pCANTAB5E-scFv plasmid was digested into two fragments by Sfi I and Not I: 750 bp of scFv and 4472 bp of pCANTAB-5E vector d The phage-ELISA showed that after three rounds of panning by H-p21Ras, K-p21Ras and N-p21Ras antigens, in turn, the number of output phages (unbound phages) was constant

at 1 × 10 6 PFU/ml, whereas the number of input phage was constant at 1 × 10 9 PFU/ml

Fig 3 Bioinformatics and immunoreactivity of the anti-p21Ras scFv a Amino acid sequence of anti-p21Ras scFv with appropriate regions for framework and CDR residues, differing from the previously reported Y13-159 scFv and Y13-238 scFv b 3D model of anti-p21Ras scFv generated

by bioinformatics Variable heavy chain (V H ) and light chain (V L ) regions of the antibody were connected by a single 15-amino acid linker (L, green).

c Western blot detected anti-p21Ras scFv antibody-specific binding with p21Ras protein in the cells, where strong immunoreactivity was found in p21Ras-overexpressing tumour cell lines; however, weak immunoreactivity was observed in the normal cell line KMB17 with low p21Ras expression.

d Immunohistochemistry revealed that anti-p21Ras scFv antibody exhibited strongly positive staining in human tumour cell lines and primary solid tumour tissues

Table 3 Immunoreactivity of KGHR1-scFv antibody in human tumour tissues and cell lines

a

50 % difference from reported p21Ras scFv

anti-bodies The KGH-R1 scFv antibody could recognize

wildtype and mutated H-p21Ras, K-p21Ras, and N-p21Ras

and exhibited strong immunoreactivity with human tumour

cell lines and primary tumours It is suggested that the scFv

may be useful for ras signalling blockage and as a

thera-peutic antibody for ras-derived tumours

Additional files

Additional file 1: The ARRIVE Guidelines Checklist (DOC 694 kb)

Abbreviations

scFv: Single chain fragment variable; kD: KiloDalton; GTP: Guanosine

triphosphate; CDS: Coding sequences; NCBI: National Center for Biotechnology

Information; IPTG: Isopropyl β-D-1-thiogalactopyranoside; SDS-PAGE: Sodium

dodecyl sulfate-polyacrylamide gel electrophoresis; mAb: Monoclonal antibody;

HAT: Hypoxanthine, aminopterin, thymidine; ELISA: Enzyme-linked

immunosorbent assay; VH: Heavy chain variable region; VL: Light chain

variable region; PBS: Phosphate buffered saline; PBST: PBS containing 0.05 %

tween-20; BSA: Albumin from bovine serum; HRP: Horseradish peroxidase;

CDR: Complementarity-determining region; FR: Framework region;

Her2: Human epidermal growth factor receptor 2.

Competing interests

The authors declare that they have no competing interests.

Authors ’ contributions

JLY contributed to the conception and design of the study DXL, SJZ, YGZ,

DJZ, LYY and HBC were involved in the acquisition of the data All authors

participated in the analysis and interpretation of the data QF drafted the

manuscript JLY contributed to the revision of the manuscript JLY contributed

to the supervision of the study All authors read and approved the final

manuscript.

Acknowledgements

This work was supported by the grants from the National Natural Science

Foundation of China (No 30872994 and No 81460464) and the Scientific and

Technological key project of Yunnan Province (No 2006SG11).

Author details

1

Department of Pathology, Kunming General Hospital/Kunming Medical

University, Kunming 650032, Yunnan Province, China 2 Department of

Molecular Biology, Kunming General Hospital/Kunming Medical University,

Kunming 650032, Yunnan Province, China.

Received: 11 June 2015 Accepted: 14 February 2016

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